CN112266420A

CN112266420A - Plant efficient cytosine single-base editor and construction and application thereof

Info

Publication number: CN112266420A
Application number: CN202011189809.5A
Authority: CN
Inventors: 刘耀光; 祝钦泷; 曾栋昌; 刘涛利
Original assignee: South China Agricultural University
Current assignee: South China Agricultural University
Priority date: 2020-10-30
Filing date: 2020-10-30
Publication date: 2021-01-26
Anticipated expiration: 2040-10-30
Also published as: CN112266420B

Abstract

The invention provides a plant efficient cytosine single-base editor and construction and application thereof. The editor comprises a SpCas9 variant fusion protein PeCBE-NG, and sequentially comprises 1 nuclear localization bpNLS functional element, 1 deaminase eCBE functional element, 1 SpCas9 variant Cas9N-NG coding sequence, 2 serial uracil glycosylase inhibiting protein UGI functional elements and 1 nuclear localization bpNLS functional element from the N end to the C end. Compared with the existing plant cytosine single base editor, the cytosine single base editor constructed by the invention has the characteristics of high efficiency, no target sequence preference, moderate editing window, low self-targeting sgRNA efficiency, low off-target efficiency and wide-target recognition of NG-PAM, is beneficial to the research of plant gene functions and the development of crop genetic improvement, and has wide application prospects in the aspects of plant single base replacement, saturation mutation screening and the like.

Description

Plant efficient cytosine single-base editor and construction and application thereof

Technical Field

The invention belongs to the technical field of plant biology, and particularly relates to a plant efficient cytosine single-base editor and construction and application thereof.

Background

Important agronomic traits of crops are often determined by point mutations or Single Nucleotide Polymorphisms (SNPs) of key genes. Although CRISPR/Cas 9-mediated genome editing can cause Double Strand Breaks (DSBs) at the target site by means of non-homologous end repair (NHEJ) to knock out the target gene efficiently, it is still more difficult to insert and replace fragments at specific sites precisely by the HDR pathway due to the inefficiency of the Homologous Direct Repair (HDR) pathway in plants and the inefficiency of DNA donor template delivery to cells (Chen et al, 2019, Annual Review of Plant Biology,70: 667-. However, Cytosine Base Editors (CBEs) and Adenine Base Editors (ABEs) (Komor et al, 2016, Nature,533: 420-424; Rees and Liu, 2018, Nature Reviews Genetics,19:770-788) developed based on the CRISPR/Cas system achieve base substitution of C-T (G-A) or A-G (T-C), respectively, without the need for a donor template and without causing double-stranded DNA breaks. Therefore, the single-base editing technology is an effective strategy for the research of plant gene functions and the genetic improvement of crops.

Currently, various research teams have developed a number of different CBEs systems by fusing cytosine deaminases (e.g., rAPOBEC1, PmCDA1, and hA3A) from different sources with Cas9 nickase variants (Cas9n, containing the D10A amino acid variation). Early versions of CBE systems, for example: BE1, BE2, BE3 and BE4 have low editing efficiency (about 0-30% on average) in Plant cells (Ren et al, 2017, Science China Life Sciences,60: 516-. New versions of CBE like BE4max were generated using codons optimized by the ancestral nodes Anc689 cytosine deaminase, bpNLS and GenScript company in the APOBEC family, although the editing efficiency in plants was further improved (on average about 50%) (Wang et al, 2019, Plant Biotechnology Journal,17:1697-The preference of rAPOBEC1 itself for target sequence environment makes it easier to edit TC、CCSites (underlined is the base edited) with little editing of GCTherefore, the editing effect is extremely unstable. Fusing PmCDA1 deaminase activating the induced cytosine deaminase (AID) family with Cas9n, the resulting Target-AID editing system is somewhat more efficient (about 40% on average) than BE3 and BE4, and the editing window is expanded (C)₂-C₁₂) (Wu et al, 2019, Frontiers in Genetics,10: 379; zhong et al, 2019, Molecular Plant,12: 1027-1036). In addition, by fusing human APOBEC3A deaminase (hA3A) and Cas9n, the resulting hA3A-PBE also improved the editing efficiency of the plant genome to some extent (approximately 40% on average), in particular GCEfficiency of target site replacement (Zong et al, 2018, Nature Biotechnology,36: 950-. Although great efforts have been made to improve the base editing system of plants, the current CBE editing system still has the problems of low activity in plants, strong preference for target sequence environment and the like. In addition, since most CBE systems are fused with Cas9n recognizing NGG-PAM, the selectivity of genome targeting is reduced, and these factors greatly limit the wide application of CBE in plant systems.

In summary, the development of a novel plant cytosine single base editor suitable for plant with high efficiency, non-sequence preference and wide targeting is an important direction for gene editing technology optimization.

Disclosure of Invention

The single base editing efficiency of plant cytosine is low, the target sequence preference of common CBEs is strong, target selection is limited by NGG-PAM, and target base C can not be successfully edited into T, so that the wide application of CBEs in plants is limited.

The invention aims to provide a plant efficient cytosine single-base editor.

The invention mainly aims to provide a method for constructing the efficient cytosine single-base editor.

The invention mainly aims to provide the application of the plant efficient cytosine single-base editor.

The purpose of the invention is realized by the following technical scheme:

a SpCas9 variant fusion protein, designated PeCBE-NG, comprising a cytosine deaminase, eCBE, a SpCas9 variant, Cas9n-NG, and a uracil glycosylase inhibitor protein (UGI).

The amino acid sequence of the cytosine deaminase eCBE is shown in SEQ ID NO. 1; the amino acid sequence of the SpCas9 variant Cas9n-NG is shown as SEQ ID NO. 2; the amino acid sequence of the uracil glycosylase inhibitor protein is shown in SEQ ID NO. 3.

Preferably, the SpCas9 variant fusion protein further comprises one or more of the following sequences: linker (Linker), Nuclear Localization Signal (NLS), and amino acid residues or amino acid sequences introduced for the construction of fusion proteins, for the promotion of expression of recombinant proteins, for the obtainment of recombinant proteins which are automatically secreted into the nucleus of the host cell, or for the facilitation of purification of recombinant proteins.

The linker may comprise any sequence that does not interfere with the function of the fusion protein, preferably a flexible linker.

The amino acid sequence of the nuclear localization signal is shown as SEQ ID NO. 4.

The number of the nuclear localization signals is preferably two, and the nuclear localization signals are respectively fused at the N end and the C end of the SpCas9 variant fusion protein.

Preferably, the number of the uracil glycosylase inhibitor proteins is two, and the two uracil glycosylase inhibitor proteins are connected in series through a linker.

More preferably, the SpCas9 variant fusion protein comprises, in order from N-terminus to C-terminus, 1 nuclear localization signal, 1 cytosine deaminase, ebe, 1 SpCas9 variant Cas9N-NG, 2 tandem uracil glycosylase inhibitor proteins, and 1 nuclear localization signal.

Most preferably, the complete amino acid sequence of the SpCas9 variant fusion protein is shown in SEQ ID No. 5. It can be obtained directly by chemical synthesis method or by gene engineering method.

A polynucleotide sequence encoding the SpCas9 variant fusion protein described above.

Preferably, the polynucleotide sequence is shown in SEQ ID NO. 6. The sequence is obtained by optimizing according to the rice codon.

A plant efficient cytosine single base editor comprises a polynucleotide sequence for coding the SpCas9 variant fusion protein, and is obtained by integrating the polynucleotide sequence for coding the SpCas9 variant fusion protein on a plant transformation vector.

Preferably, the plant transformation vector is a binary expression vector, including but not limited to pCAMBIA1300 and a vector modified on the basis of pCAMBIA1300, such as pYLCRISPR/Cas9Pubi-H, and the polynucleotide sequence can be inserted between the enzyme cutting sites Pst I and BamH I of the vector.

The construction method of the plant efficient cytosine single-base editor comprises the following steps:

s1, respectively synthesizing a gene segment 1 for coding NLS-eCBE-linker1 and a gene segment 2 for coding linker2-UGI-linker 3-UGI-NLS, adding one enzyme cutting site at the 5 'end of the gene segment 1 through PCR reaction, and adding the other enzyme cutting site at the 3' end of the gene segment 2; cloning to obtain a gene fragment 3 for encoding Cas9 n-NG;

s2, connecting the C end of the gene fragment 1 with the enzyme cutting site with the N end of the gene fragment 3 by an overlapping PCR technology to obtain a gene fragment 4 of a fusion protein NLS-eCBE-linker1-Cas 9N-NG; then connecting the C end of the gene segment 4 with the N end of the gene segment 2 with the enzyme cutting site to obtain a gene segment 5 of a fusion protein NLS-eCBE-linker1-Cas9N-NG-linker2-UGI-linker 3-UGI-linker-NLS;

s3, inserting the gene fragment 5 between two corresponding enzyme cutting sites of a vector pYLCRISPR/Cas9Pubi-H, transforming host bacteria, extracting positive plasmids, sequencing and obtaining the stable plant efficient cytosine single-base editing system.

Preferably, the insertion of the gene fragment 5 into the vector in step S3 is performed by Gibson assembly technique.

Preferably, the host bacterium described in step S3 is Escherichia coli Top 10F'.

The SpCas9 variant fusion protein, the polynucleotide sequence or the plant efficient cytosine single base editor can be applied to plant genetic engineering. In particular to the application in single base editing of plant genome. Such plants include, but are not limited to, monocots, particularly rice.

Preferably, the application specific operation is as follows:

(1) determining a target site of a gene to be edited, and designing and synthesizing an sgRNA expression cassette element according to the target site;

(2) integrating the sgRNA expression cassette element to the plant efficient cytosine single base editor to obtain a target gene cytosine base substitution vector;

(3) and transforming the target gene cytosine base substitution vector into a host cell, and screening to obtain a corresponding cytosine base substitution cell.

The invention is suitable for efficient cytosine base editor pYL-PeCBE-NG with non-sequence preference, suitable editing window, low off-target efficiency and wide target of plants, and the expression cassette of the SpCas9 variant fusion protein PeCBE-NG sequentially comprises a plant high-expression constitutive promoter Ubi, a SpCas9 variant fusion protein PeCBE-NG (nuclear localization signal bpNLS, eCBE cytosine deaminase, flexible connecting sequence linker1 (encoding 32aa), a Cas9n-NG mutant, a flexible connecting sequence linker2 (encoding 9aa), uracil glycosylase inhibitor protein UGI, flexible connecting sequence linker3 (encoding 9aa), uracil glycosylase inhibitor protein UGI, nuclear localization signal bpNLS) and a terminator Tnos sequence from 5 'end to 3' end. The base sequence of the PeCBE-NG encoding SpCas9 variant fusion protein is optimized and synthesized by King Share (Wuhan, China) according to the rice codon preference.

The pYL-PeCBE-NG cytosine single base editing system constructed by the invention has high replacement efficiency of C-T, no target sequence preference and a main editing activity window at C₃-C₈Meanwhile, the editing efficiency of self-targeting T-DNA is low, and the off-target efficiency is also low. Overcomes the defects of low editing efficiency, strong target preference and the like of the traditional BE3 and BE4 editors using the deaminase rAPOBEC1, and is more beneficial to the research of plant gene functions and the genetic improvement of crops.

Materials such as plasmid vector of cytosine single base editing system containing SpCas9 variant fusion protein PeCBE-NG and application of the PeCBE-NG cytosine single base editor in genetic engineering are both within the protection scope of the invention.

More particularly, the pYL-PeCBE-NG cytosine single base editor system can realize efficient C-T single base substitution, and is particularly suitable for gene function research such as gene function screening, large-scale saturation mutation, editing and regulating elements, introduction of a premature stop codon or alternative splicing and the like, and genetic improvement of crops.

The invention has the following beneficial effects:

the plant CBE single-base editing system provided by the invention has the advantages of high efficiency, no target point sequence preference, wide editing window, low self-targeting sgRNA efficiency, less non-C-T byproducts, low off-target efficiency, wide targeting range and capability of multi-target editing. Compared with other editing systems existing at present, the editing of pYL-PeCBE-NG is more advantageous.

Drawings

FIG. 1 is a schematic diagram of the structure of the cytosine single base editor pYL-PeCBE-NG vector of the present invention.

FIG. 2 is a graph of pYL-PeCBE-NG versus BE4 editor pYL-rAC1-NG editing efficiency comparison results; wherein, A is a map of pYL-PeCBE-NG and pYL-rAC1-NG with different target sites, B is the comparison result of the editing efficiency of pYL-PeCBE-NG and pYL-rAC1-NG in 9 target points (the number of the mutated plants is the total number of the plants in parentheses, and PAM in the target points is underlined and bolded and highlighted).

FIG. 3 is a graph of the results of a comparative analysis of target preference and mutation type for pYL-PeCBE-NG and pYL-rAC 1-NG; wherein, A is the result of the average editing efficiency of NGG and NG targets counted from 9 tested targets, and B is C edited stably₃-C₈Analysis of G in all mutations occurring in the Window IntervalC，AC，TC，CC(C is the base edited), C is the result of mutation type analysis (Ho means homozygous mutation; He means heterozygous mutation; Transns means transitions), Index means insertions or deletions meansExcept).

FIG. 4 is a graph comparing the activity windows of pYL-PeCBE-NG and pYL-rAC 1-NG.

FIG. 5 is a graph of the results of the pYL-PeCBE-NG self-targeted editing efficiency analysis.

FIG. 6 is a graph of pYL-PeCBE-NG off-target efficiency analysis results (bases of On-target sequence differences are underlined and highlighted in bold, PAM at the target site is highlighted in bold).

Detailed Description

The present invention will be further described with reference to the following specific examples and drawings, which are not intended to limit the invention in any manner.

Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated. The test methods used in the following examples are all conventional molecular biological methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.

EXAMPLE 1 construction of plant cytosine Single base editor pYL-PeCBE-NG

The protein sequences of deaminase eCBE, 2 XUGI and bpNLS are directly synthesized by a WuGeneHan company according to sequences published by Thuronyi and the like (Thuronyi et al, 2019, Nature Biotechnology,37:1070-1079) and according to bpNLS-eCBE-linker1 and linker2-UGI-linker3-UGI-bpNLS two fragments according to the preference of rice codons (nucleotides of bpNLS-eCBE-linker1 are shown as bases at positions 4-636 of SEQ ID NO.6, and nucleotides of linker2-UGI-linker 3-UGI-NLbps are shown as bases at positions 4738-5361 of SEQ ID NO. 6). The optimally synthesized Cas9n-NG variant (Zeng et al, 2020, Plant Biotechnology Journal,18:1348-1350) already available to the applicant (shown as bases 637-4737 of SEQ ID NO. 6) was used directly for fusion. The optimally synthesized bpNLS-eCBE-linker1 and linker2-UGI-linker3-UGI-bpNLS are respectively connected to two sides of Cas9n-NG through Overlaping PCR to form complete fused PeCBE-NG, and then cloned between Pst I and BamH I of a binary vector pYLCRISPR/Cas9Pubi-H (Ma et al, 2015, Molecular Plant,8: 1274-. The BE4 editor pYL-Cas9n-NG-CBE (pYL-rAC 1-NG) (Zeng et al, 2020, Plant Biotechnology Journal,18: 1348-.

TABLE 1 primers for basic vector engineering for pYL-PeCBE-NG

Name of the lead	Primer sequences (5 '-3')
		F-PeCBE-NG-1	CTCACCCTGTTGTTTGGTGTTACTTctgcagATGAAGAGGACAGCCG(SEQ ID NO.7)
R-PeCBE-NG-1	GCCGATGGAGTACTTCTTGTCGGAGCCGCCGGAGCTGCCACCAG(SEQ ID NO.8)
		F-PeCBE-NG-2	CTCCGGCGGCTCCGACAAGAAGTACTCCATC(SEQ ID NO.9)
R-PeCBE-NG-2	CCAGATCCACCAGAATCACCACCAAGCTGGGA(SEQ ID NO.10)
		F-PeCBE-NG-3	CAGCTTGGTGGTGATTCTGGTGGATCTGGCGGAAG(SEQ ID NO.11)
R-PeCBE-NG-3	CAAATGTTTGAACGATCGGGAggatccTAGACCTTGCGCTTCTTC(SEQ ID NO.12)

The specific procedure is as follows:

1. construction of plant efficient Cytosine base editor pYL-PeCBE-NG

An F-PeCBE-NG-1/R-PeCBE-NG-1(SEQ ID NO.7 and SEQ ID NO.8) primer is used, bpNLS-eCBE-linker1 synthesized by a company is used as a template, and a bpNLS-eCBE-linker1 fragment with a Ps tI enzyme cutting site is obtained through amplification and is named as a fragment M.

PCR System (15. mu.l): 2 x Phanta Max Buffer 7.5. mu.l, 10mmol/L dNTPs Mix 0.35. mu.l, Phanta Max polymerase0.35. mu.l, bpNLS-eCBE-linker 110 NG, 10. mu. mol/L F-PeCBE-NG-10.35. mu.l, 10. mu. mol/L R-PeCBE-NG-10.35. mu.l, ddH₂O make up to 15. mu.l.

PCR procedure: pre-denaturation at 95 ℃ for 2min, 28 PCR cycles (95 ℃ for 10s,56 ℃ for 15s,72 ℃ for 30s), extension at 72 ℃ for 2 min.

And (3) amplifying to obtain a Cas9n-NG fragment by using F-PeCBE-NG-2/R-PeCBE-NG-2(SEQ ID NO.9 and SEQ ID NO.10) primers and using pYL-Cas9n-NG-CBE plasmid as a template.

PCR System (15. mu.l): 2 x Phanta Max Buffer 7.5. mu.l, 10mmol/L dNTPs Mix 0.35. mu.l, Phanta Max polymerase0.35. mu.l, pYL-Cas9n-NG-CBE 10NG, 10. mu. mol/L F-PeCBE-NG-20.35. mu.l, 10. mu. mol/L R-PeCBE-NG-20.35. mu.l, ddH₂O make up to 15. mu.l.

PCR procedure: pre-denaturation at 95 ℃ for 2min, 28 PCR cycles (95 ℃ for 10s,56 ℃ for 15s,72 ℃ for 4min), extension at 72 ℃ for 5 min.

The DNA fragment of the linker2-UGI-linker3-UGI-bpNLS with BamHI cleavage site was amplified using F-PeCBE-NG-3/R-PeCBE-NG-3(SEQ ID NO.11 and SEQ ID NO.12) primers and linker2-UGI-linker3-UGI-bpNLS synthesized by the company as a template, and named fragment N.

PCR System (15. mu.l): 2 x Phanta Max Buffer 7.5. mu.l, 10mmol/L dNTPs Mix 0.35. mu.l, Phanta Max Polymer 0.35. mu.l, linker2-UGI-linker3-UGI-bpNLS 10NG, 10. mu. mol/L F-PeCBE-NG-30.35. mu.l, 10. mu. mol/L R-PeCBE-NG-30.35. mu.l, ddH₂O make up to 15. mu.l.

A bpNLS-eCBE-linker1-Cas9n-NG fusion DNA fragment was amplified using F-PeCBE-NG-1/R-PeCBE-NG-2(SEQ ID NO.7 and SEQ ID NO.10) primers with the first round of amplified eCBE fragment M and Cas9n-NG fragment as templates.

PCR System (15. mu.l): 2 x Phanta Max Buffer 7.5. mu.l, 10mmol/L dNTPs Mix 0.35. mu.l, Phanta Max polymerase0.35. mu.l, first round amplified eCBE fragment M and Cas9n-NG 0.1. mu.l, 10. mu. mol/L F-PeCBE-NG-10.35. mu.l, 10. mu. mol/L R-PeCBE-NG-40.35. mu.l, ddH₂O make up to 15. mu.l.

PCR procedure: pre-denaturation at 95 ℃ for 2min, 28 PCR cycles (95 ℃ for 10s,56 ℃ for 15s,72 ℃ for 4min), extension at 72 ℃ for 2 min.

And (3) amplifying bpNLS-eCBE-linker1-Cas9N-NG-linker2-UGI-linker3-UGI-linker-bpNLS fusion fragment (eCBE-Cas 9N-NG-2 XUGI fusion fragment for short) by using F-PeCBE-NG-1/R-PeCBE-NG-3(SEQ ID NO.7 and SEQ ID NO.12) primers and taking the eCBE-Cas9N-NG fusion DNA fragment amplified in the second round and the first round amplification fragment N as templates.

PCR System (50. mu.l): 2 x Phanta Max Buffer25. mu.l, 10mmol/L dNTPs Mix 1.0. mu.l, Phanta Max Polymerase 1.0. mu.l, linker2-UGI-linker3-UGI-bpNLS fragment N amplified in the first round and eBE-Cas 9N-NG fusion DNA fragment amplified in the second round were 1.0. mu.l, 10. mu.mol/L F-PeCBE-NG-11.0. mu.l, 10. mu.mol/L R-PeCBE-NG-41.0. mu.l, ddH₂O make up to 50. mu.l.

PCR procedure: pre-denaturation at 95 ℃ for 2min, 28 PCR cycles (95 ℃ for 10s,56 ℃ for 15s,72 ℃ for 4.5min), extension at 72 ℃ for 2 min.

The PCR product of the amplified bpNLS-eCBE-Linker1-Cas9n-NG-Linker2-UGI-Linker3-UGI-bpNLS fusion DNA fragment was purified with Genstar purification kit. pYRCISPR/Cas 9Pubi-H (Ma et al, 2015, Molecular Plant,8: 1274-: 10 × fast digest buffer, Pst I0.5 μ l, BamH I0.5 μ l, pYLCRISPR/Cas9 Pubi-H300 ng, ddH₂O to 10. mu.l, reaction at 37 ℃ for 1h, gel recovery of the support backbone for Gibson assembly (NEB # E5510S): 2 x Mix 5. mu.l, eCBE-Cas9n-NG-2 x UGI fusion fragment 60NG, gel recovery vector backbone 90NG, ddH₂Make up to 10. mu.l of O, and react at 50 ℃ for 50 min. Mu.l of the ligation product of Gibson was taken and E.coli Top 10F' was electrically transformed, in kanamycinResistant (Kana) LB plates were screened for single transformants. And the positive clones were sequenced to obtain pYL-PeCBE-NG basic vector plasmid.

Example 2 pYL-PeCBE-NG has higher efficiency in cytosine base editing

Referring to the earlier published literature (Ma et al 2015, Molecular Plant 8: 1274-1284; Ma and Liu,2016, Current Protocols in Molecular Biology 115: 31.6.1-31.6.21; Zengdongchang et al 2018, China science: Life sciences 48:783-794), small nuclear RNA gene promoters (OsU6a, OsU6b, OsU6c and OsU3) are respectively constructed to drive sgRNA expression cassettes of different targets, pYL-PeCBE-NG binary vectors (Ma et al 2015, Molecular Plant 8:1274-1284) are inserted by using a 'gold Gate assembly and Golden Gate' mode, rice is transformed, and the transformants are sequenced to analyze the editing efficiency of pYL-PeCBE-NG. The specific operation is as follows:

target primer design of T1-T9

The CRISPR-GE webpage (http:// skl. scau. edu. cn /) (Xie et al, 2018, Molecular plant,11: 720-.

Overlapping PCR splicing of sgRNA expression cassettes from T1 to T9

Two rounds of PCR were performed to obtain sgRNA expression cassettes driven by small RNA promoters with Bsa I cleavage sites on both sides, according to the published literature (Ma et al 2015, Molecular Plant 8: 1274-.

TABLE 2 first round PCR target primer gR-T # and U # -T # sequences

Name of the lead	Sequence (5 '- -3')
		gRT1	ACCCCCCCACAGGCTCGCGAgttttagagctagaaat(SEQ ID NO.13)
OsU6aT1	TCGCGAGCCTGTGGGGGGGTCggcagccaagccagca(SEQ ID NO.14)
		gRT2	TCGTCGGCGGCGATGGTGAgttttagagctagaaat(SEQ ID NO.15)
OsU6aT2	TCACCATCGCCGCCGACGACggcagccaagccagca(SEQ ID NO.16)
		gRT3	ACCGCCACCGTCGTCGCCAAgttttagagctagaaat(SEQ ID NO.17)
OsU6bT3	TTGGCGACGACGGTGGCGGTCaacacaagcggcagc(SEQ ID NO.18)
		gRT4	TGCATGCATGCACCCATGCgttttagagctagaaat(SEQ ID NO.19)
OsU3T4	GCATGGGTGCATGCATGCATgccacggatcatctgc(SEQ ID NO.20)
		gRT5	ATCTCTGCACTGAATTGAATgttttagagctagaaat(SEQ ID NO.21)
OsU6aT5	ATTCAATTCAGTGCAGAGATCggcagccaagccagca(SEQ ID NO.22)
		gRT6	TCCACCATGCACCACGACGTgttttagagctagaaat(SEQ ID NO.23)
OsU6aT6	ACGTCGTGGTGCATGGTGGACggcagccaagccagca(SEQ ID NO.24)
		gRT7	AGCTCAAGCTCCGCGCCGCgttttagagctagaaat(SEQ ID NO.25)
OsU6bT7	GCGGCGCGGAGCTTGAGCTCaacacaagcggcagc(SEQ ID NO.26)
		gRT8	ATCAGCGACCGGATCTCCCCgttttagagctagaaat(SEQ ID NO.27)
OsU6bT8	GGGGAGATCCGGTCGCTGATCaacacaagcggcagc(SEQ ID NO.28)
		gRT9	CATTCTCCCAGTTCTTCGCgttttagagctagaaat(SEQ ID NO.29)
OsU3T9	GCGAAGAACTGGGAGAATGTgccacggatcatctgc(SEQ ID NO.30)
		U-F	CTCCGTTTTACCTGTGGAATCG(SEQ ID NO.31)
gR-R	CGGAGGAAAATTCCATCCAC(SEQ ID NO.32)

In the first round of PCR, a designed U # -T #/gR-T # primer (SEQ ID NO. 13-SEQ ID NO.30, Table 2) is used for introducing a target point sequence into the downstream of OsU6/OsU3 promoter and the upstream of sgRNA sequence. In a PCR system, a U-F primer (SEQ ID NO.31, table 2) is paired with a gR-T # primer, and a promoter sequence containing a target spot is obtained through PCR amplification; and pairing a gR-R primer (SEQ ID NO.32, table 2) with a U # -T # primer, and carrying out PCR amplification to obtain a sgRNA sequence containing a target spot. PCR System (20. mu.l): 2 × Phanta Max Buffer 10.0 μ L, 10mmol/L dNTPs Mix 0.4 μ L, Phanta Max Polymerase0.3 μ L, pYLgRNA-OsU6/3 (containing promoter and sgRNA plasmid) (Ma et al, 2015, Molecular Plant,8: 1274. sub.1284) 3ng, 10 μmol/L U-F0.4 μ L, 10 μmol/L gR-T #0.2 μ L, 10 μmol/L gRNA-R0.4 μ L, 10 μmol/L U # -T #0.2 μ L, ddH2O to 20 μ L. PCR procedure: pre-denaturation at 95 ℃ for 1min, 28 PCR cycles (95 ℃ for 10s,58 ℃ for 15s,72 ℃ for 20s), extension at 72 ℃ for 1 min.

Table 3 construction of universal primers for multiple sgRNA expression cassettes

Note 1: bsa I cleavage ends were designed as non-palindromic sequences, which resulted in efficient ligation (Golden Gate ligation).

Note 2: if more than 8 sgRNA expression cassettes were ligated, it was necessary to design by itself more sets of Pgs and Pps primers, each set containing complementary non-palindromic Bsa I cleaved ends.

Table 4 second round PCR primer combinations to assemble different numbers of sgRNA expression cassettes

Second round PCR, splicing Small RNA promoters with second round PCR primers Pps and Pgs (SEQ ID NO. 33-SEQ ID NO.48, tables 3 and 4)The sgRNA expression cassette was driven and a Bsa I cleavage site was added to both sides of the PCR product. And constructing 1 sgRNA expression cassette vector with 4 target points and 5 target points respectively by taking T1-T4 as a group and T5-T9 as a group. PCR System (50. mu.l): 2 x Phanta Max buffer25.0 μ L, 10mmol/L dNTPs Mix 1.0 μ L, Phanta Max Polymerase 1.0 μ L, 10 x diluting the previous round of PCR product 1.0 μ L, 4 target respectively used primers, 10 μmol/L Pps-L/Pgs-2(T1), Pps-2/Pgs-3(T2), Pps-3/Pgs-4(T3), Pps-4/Pgs-R (T4) each 1.0 μ L; primers used for 5 targets, 10. mu. mol/L Pps-L/Pgs-2(T1), Pps-2/Pgs-3(T2), Pps-3/Pgs-4(T3), Pps-4/Pgs-5(T4), Pps-5/Pgs-R (T5) are 1.0. mu.l each, ddH₂O to 50. mu.l, and the PCR procedure was the same as the first round of PCR described above. The second round PCR product was purified using the gentar purification kit.

3. Construction of sgRNA expression cassette knockout vector containing different target points

The method comprises the steps of respectively assembling T1-T4 and T5-T9 by using a Bsa I enzyme digestion and connection-based 'gold gate' cloning method and in a 'side-cutting side-connecting' mode (Ma and Liu,2016, Current Protocols in Molecular Biology,115: 31.6.1-31.6.21; Zengdongchang et al, 2018, China science: Life sciences, 48: 783-; two groups of sgRNA expression cassettes driven by small nuclear RNA promoters were cloned into binary vector pYL-PeCBE-NG, respectively (FIG. 2A). 15 μ l reaction: 10 x CutSmart Buffer 1.5 μ L, 10mmol/L ATP 1.5 μ L, pYL-PeCBE-NG plasmid 80-100 NG, purified sgRNA expression cassette 10-15 NG, Bsa I-HF 10U, T4 DNA ligase 35U, ddH2O make up to 15 μ L. Carrying out enzyme digestion and ligation reaction by using a PCR instrument in a temperature-variable circulation manner: 10min at 37 ℃ followed by 10-12 cycles (5 min at 37 ℃, 3min at 10 ℃, 5min at 20 ℃); finally 3min at 37 ℃. After dialysis of the ligation products, electrical stimulation was performed to transform into DH10B cells, selection was performed on Carla-resistant (Kan) LB plates, with the primer pair SP-L1/SP-R (SEQ ID NO.49 and SEQ ID NO.50, Table 3), colony PCR was performed according to the literature (Ma and Liu,2016, Current Protocols in Molecular Biology,115: 31.6.1-31.6.21; Zeemann Changchang et al, 2018, China science: Life sciences, 48: 783. times. 794), positive clones were selected, and finally sequencing was performed with the primer SP-L1(SEQ ID NO. 49).

4, pYL-PeCBE-NG has higher editing efficiency.

Using Agrobacterium tumefaciensTransforming the callus of rice (japonica rice middle flower 11), transforming the rice callus with the 4-target and 5-target vectors containing sgRNA driven by two different small RNA promoters T1-T4 and T5-T9 (figure 2A), extracting T₀Leaf DNA of the generation-transformed plants was used as a template, and the DNA fragment of the single-base editing target site was amplified using amp and SEQ (SEQ ID NO.51 to SEQ ID NO.77, Table 5), subjected to Sanger sequencing directly, and the editing efficiency of pYL-rAC1-NG and pYL-PeCBE-NG was counted and compared by aligning the sequencing results with the reference sequence. The results showed that the average editing efficiency of pYL-PeCBE-NG was the highest (62.6%), with the editing efficiency at the T7 target being as high as 86.3% (FIG. 2B).

5, pYL-PeCBE-NG also has higher editing efficiency in NG targets, and no target sequence preference with fewer by-products.

Cas9-NG expands the editing range of genome, but the cleavage editing efficiency is reduced to a certain extent compared with that of the traditional SpCas9 (Zeng et al, 2020, Plant Biotechnology Journal,18: 1348) in order to investigate whether the pYL-PeCBE-NG single-base editor can realize efficient editing at the NG target, we counted the average editing efficiency of pYL-PeCBE-NG at the NGG target and the NG target from 9 targets, and the result shows that the average editing efficiency of pYL-PeCBE-NG at the NGG target is slightly lower than that of the NG target, but the higher editing efficiency is maintained at the NG target (FIG. 3A). Based on the sequencing results of the T0 generation, we counted all vectors tested at C₃-C₈On the active window GC、AC、TCAnd CCThe motifs were averaged for editing efficiency and their base bias was analyzed. Statistical results show that rAC1 deaminase rAC1-NG with BE4 as a carrier structure has the preference of severe sequence environment at TC(8.8%) and CC(5.2%) had low editing efficiency, and on GCAnd ACNo editing occurred at any of the 9 targets (FIG. 3B). And pYL-PeCBE-NG eliminates the preference of target point sequence environment and has higher editing efficiency. Furthermore, the edited product of pYL-PeCBE-NG was predominantly homozygous and heterozygous mutated, whereas the byproduct of non-C-T editing was only 2.9% (FIG. 3C).

Table 5T 0 generation transformed plant target amplification and sequencing primer

6, pYL-PeCBE-NG has a moderate edit window

According to the sequencing result of the T0 generation, the statistics that 9 targets in T1-T9 are at C_-2～C₁₅The average editing efficiency of C-T in each site is plotted as an editing activity window. The results show that pYL-PeCBE-NG has a relatively modest edit window (C)₃-C₈) (FIG. 4). The deaminase with a moderate editing window is matched with the wide-target Cas protein, so that the application of a single-base editing technology in the genome editing of animals and plants is facilitated.

7, pYL-PeCBE-NG is inefficient in self-targeting sgRNA targets

In previous reports, Cas9-NG could recognize GTT-PAM in addition to NGG-PAM, and thus Cas9-NG could target both genomic and T-DNA own sgRNA targets (Qin et al, 2020, Nat Plants 6: 197-201). To explore the efficiency of pYL-PeCBE-NG in this study from targeting the T-DNA self sgRNA target site, T was directly amplified₀Sequencing of sgRNA targets of the generation transformed seedlings by Sanger showed that PeCBE-NG only exhibited weak sgRNA self-editing efficiency at T6 target (6.9%), whereas no editing was detected at other targets (fig. 5), with an average self-editing efficiency of 9 targets being much lower than that of the known SpCas9-NG of more than 50%.

The off-target efficiency of 8, pYL-PeCBE-NG is also low

A CRISPR-GE webpage (http:// skl.scau.edu.cn /) (Xie et al, 2018, Molecular plant,11: 720-. And (3) carrying out PCR amplification on potential off-target sites by using the resistant callus genome DNA as a template for high-throughput sequencing analysis. The results show that PeCBE-NG found no off-target at candidate off-target sites that did not match at 2 and 3 bases, except for the occurrence of off-target detected at potential off-target sites that did not match at 1 base (FIG. 6). pYL-PeCBE-NG is demonstrated to have low sgRNA-induced off-target efficiency.

In conclusion, compared with the previous CBE editing system (such as the previous BE4 editor pYL-rAC1-NG of the applicant), the novel efficient plant cytosine base editor pYL-PeCBE-NG has the advantages of lower off-target efficiency and the like, and can BE widely used for operations such as crop gene function screening, large-scale saturation mutation, regulation and control element editing, early stop codon introduction or alternative splicing and the like.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Sequence listing

<110> southern China university of agriculture

<120> plant efficient cytosine single-base editor and construction and application thereof

<160> 50

<170> SIPOSequenceListing 1.0

<210> 1

Phe Glu Arg Asn Tyr Asp Pro Arg Glu Leu Arg Lys Glu Thr Tyr Leu

1 5 10 15

Leu Tyr Glu Ile Lys Trp Gly Lys Ser Gly Lys Leu Trp Arg His Trp

20 25 30

Cys Gln Asn Asn Arg Thr Gln His Ala Glu Val Tyr Phe Leu Glu Asn

35 40 45

Ile Phe Asn Ala Arg Arg Phe Asn Pro Ser Thr His Cys Ser Ile Thr

50 55 60

Trp Tyr Leu Ser Trp Ser Pro Cys Ala Glu Cys Ser Gln Lys Ile Val

65 70 75 80

Asp Phe Leu Lys Glu His Pro Asn Val Asn Leu Glu Ile Tyr Val Ala

85 90 95

Arg Leu Tyr Tyr Pro Glu Asn Glu Arg Asn Arg Gln Gly Leu Arg Asp

100 105 110

Leu Val Asn Ser Gly Val Thr Ile Arg Ile Met Asp Leu Pro Asp Tyr

115 120 125

Asn Tyr Cys Trp Lys Thr Phe Val Ser Asp Gln Gly Gly Asp Glu Asp

130 135 140

Tyr Trp Pro Gly His Phe Ala Pro Trp Ile Lys Gln Tyr Ser Leu Lys

145 150 155 160

Leu

<210> 2

Asp Lys Lys Tyr Ser Ile Gly Leu Ala Ile Gly Thr Asn Ser Val Gly

1 5 10 15

Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe Lys

20 25 30

Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile Gly

35 40 45

Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu Lys

50 55 60

Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys Tyr

65 70 75 80

Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser Phe

85 90 95

Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys His

100 105 110

Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr His

115 120 125

Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp Ser

130 135 140

Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His Met

145 150 155 160

Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro Asp

165 170 175

Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr Asn

180 185 190

Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala Lys

195 200 205

Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn Leu

210 215 220

Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn Leu

225 230 235 240

Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe Asp

245 250 255

Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp Asp

260 265 270

Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp Leu

275 280 285

Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp Ile

290 295 300

Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser Met

305 310 315 320

Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys Ala

325 330 335

Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe Asp

340 345 350

Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser Gln

355 360 365

Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp Gly

370 375 380

Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg Lys

385 390 395 400

Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu Gly

405 410 415

Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe Leu

420 425 430

Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile Pro

435 440 445

Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp Met

450 455 460

Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu Val

465 470 475 480

Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr Asn

485 490 495

Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser Leu

500 505 510

Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys Tyr

515 520 525

Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln Lys

530 535 540

Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr Val

545 550 555 560

Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp Ser

565 570 575

Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly Thr

580 585 590

Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp Asn

595 600 605

Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr Leu

610 615 620

Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala His

625 630 635 640

Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr Thr

645 650 655

Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp Lys

660 665 670

Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe Ala

675 680 685

Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe Lys

690 695 700

Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu His

705 710 715 720

Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly Ile

725 730 735

Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly Arg

740 745 750

His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln Thr

755 760 765

Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile Glu

770 775 780

Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro Val

785 790 795 800

Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu Gln

805 810 815

Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg Leu

820 825 830

Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys Asp

835 840 845

Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg Gly

850 855 860

Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys Asn

865 870 875 880

Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys Phe

885 890 895

Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp Lys

900 905 910

Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr Lys

915 920 925

His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp Glu

930 935 940

Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser Lys

945 950 955 960

Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg Glu

965 970 975

Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val Val

980 985 990

Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe Val

995 1000 1005

Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala Lys Ser

1010 1015 1020

Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe Tyr Ser Asn

1025 1030 1035 1040

Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala Asn Gly Glu Ile

1045 1050 1055

Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr Gly Glu Ile Val

1060 1065 1070

Trp Asp Lys Gly Arg Asp Phe Ala Thr Val Arg Lys Val Leu Ser Met

1075 1080 1085

Pro Gln Val Asn Ile Val Lys Lys Thr Glu Val Gln Thr Gly Gly Phe

1090 1095 1100

Ser Lys Glu Ser Ile Arg Pro Lys Arg Asn Ser Asp Lys Leu Ile Ala

1105 1110 1115 1120

Arg Lys Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Val Ser Pro

1125 1130 1135

Thr Val Ala Tyr Ser Val Leu Val Val Ala Lys Val Glu Lys Gly Lys

1140 1145 1150

Ser Lys Lys Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met

1155 1160 1165

Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys

1170 1175 1180

Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr

1185 1190 1195 1200

Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala

1205 1210 1215

Arg Phe Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val

1220 1225 1230

Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser Pro

1235 1240 1245

Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys His Tyr

1250 1255 1260

Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys Arg Val Ile

1265 1270 1275 1280

Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala Tyr Asn Lys His

1285 1290 1295

Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile Ile His Leu Phe

1300 1305 1310

Thr Leu Thr Asn Leu Gly Ala Pro Arg Ala Phe Lys Tyr Phe Asp Thr

1315 1320 1325

Thr Ile Asp Arg Lys Val Tyr Arg Ser Thr Lys Glu Val Leu Asp Ala

1330 1335 1340

Thr Leu Ile His Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg Ile Asp

1345 1350 1355 1360

Leu Ser Gln Leu Gly Gly Asp

1365

<210> 3

Thr Asn Leu Ser Asp Ile Ile Glu Lys Glu Thr Gly Lys Gln Leu Val

1 5 10 15

Ile Gln Glu Ser Ile Leu Met Leu Pro Glu Glu Val Glu Glu Val Ile

20 25 30

Gly Asn Lys Pro Glu Ser Asp Ile Leu Val His Thr Ala Tyr Asp Glu

35 40 45

Ser Thr Asp Glu Asn Val Met Leu Leu Thr Ser Asp Ala Pro Glu Tyr

50 55 60

Lys Pro Trp Ala Leu Val Ile Gln Asp Ser Asn Gly Glu Asn Lys Ile

65 70 75 80

Lys Met Leu

<210> 4

Lys Arg Thr Ala Asp Gly Ser Glu Phe Glu Ser Pro Lys Lys Lys Arg

1 5 10 15

Lys Val

<210> 5

Met Lys Arg Thr Ala Asp Gly Ser Glu Phe Glu Ser Pro Lys Lys Lys

1 5 10 15

Arg Lys Val Phe Glu Arg Asn Tyr Asp Pro Arg Glu Leu Arg Lys Glu

20 25 30

Thr Tyr Leu Leu Tyr Glu Ile Lys Trp Gly Lys Ser Gly Lys Leu Trp

35 40 45

Arg His Trp Cys Gln Asn Asn Arg Thr Gln His Ala Glu Val Tyr Phe

50 55 60

Leu Glu Asn Ile Phe Asn Ala Arg Arg Phe Asn Pro Ser Thr His Cys

65 70 75 80

Ser Ile Thr Trp Tyr Leu Ser Trp Ser Pro Cys Ala Glu Cys Ser Gln

85 90 95

Lys Ile Val Asp Phe Leu Lys Glu His Pro Asn Val Asn Leu Glu Ile

100 105 110

Tyr Val Ala Arg Leu Tyr Tyr Pro Glu Asn Glu Arg Asn Arg Gln Gly

115 120 125

Leu Arg Asp Leu Val Asn Ser Gly Val Thr Ile Arg Ile Met Asp Leu

130 135 140

Pro Asp Tyr Asn Tyr Cys Trp Lys Thr Phe Val Ser Asp Gln Gly Gly

145 150 155 160

Asp Glu Asp Tyr Trp Pro Gly His Phe Ala Pro Trp Ile Lys Gln Tyr

165 170 175

Ser Leu Lys Leu Ser Gly Gly Ser Ser Gly Gly Ser Ser Gly Ser Glu

180 185 190

Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Ser Gly Gly Ser

195 200 205

Ser Gly Gly Ser Asp Lys Lys Tyr Ser Ile Gly Leu Ala Ile Gly Thr

210 215 220

Asn Ser Val Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser

225 230 235 240

Lys Lys Phe Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys

245 250 255

Asn Leu Ile Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala

260 265 270

Thr Arg Leu Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn

275 280 285

Arg Ile Cys Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val

290 295 300

Asp Asp Ser Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu

305 310 315 320

Asp Lys Lys His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu

325 330 335

Val Ala Tyr His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys

340 345 350

Leu Val Asp Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala

355 360 365

Leu Ala His Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp

370 375 380

Leu Asn Pro Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val

385 390 395 400

Gln Thr Tyr Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly

405 410 415

Val Asp Ala Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg

420 425 430

Leu Glu Asn Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu

435 440 445

Phe Gly Asn Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys

450 455 460

Ser Asn Phe Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp

465 470 475 480

Thr Tyr Asp Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln

485 490 495

Tyr Ala Asp Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu

500 505 510

Leu Ser Asp Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu

515 520 525

Ser Ala Ser Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr

530 535 540

Leu Leu Lys Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu

545 550 555 560

Ile Phe Phe Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly

565 570 575

Gly Ala Ser Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu

580 585 590

Lys Met Asp Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp

595 600 605

Leu Leu Arg Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln

610 615 620

Ile His Leu Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe

625 630 635 640

Tyr Pro Phe Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr

645 650 655

Phe Arg Ile Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg

660 665 670

Phe Ala Trp Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn

675 680 685

Phe Glu Glu Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu

690 695 700

Arg Met Thr Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro

705 710 715 720

Lys His Ser Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr

725 730 735

Lys Val Lys Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser

740 745 750

Gly Glu Gln Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg

755 760 765

Lys Val Thr Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu

770 775 780

Cys Phe Asp Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala

785 790 795 800

Ser Leu Gly Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp

805 810 815

Phe Leu Asp Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu

820 825 830

Thr Leu Thr Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys

835 840 845

Thr Tyr Ala His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg

850 855 860

Arg Arg Tyr Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly

865 870 875 880

Ile Arg Asp Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser

885 890 895

Asp Gly Phe Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser

900 905 910

Leu Thr Phe Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly

915 920 925

Asp Ser Leu His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile

930 935 940

Lys Lys Gly Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys

945 950 955 960

Val Met Gly Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg

965 970 975

Glu Asn Gln Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met

980 985 990

Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys

995 1000 1005

Glu His Pro Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu

1010 1015 1020

Tyr Tyr Leu Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp

1025 1030 1035 1040

Ile Asn Arg Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser

1045 1050 1055

Phe Leu Lys Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp

1060 1065 1070

Lys Asn Arg Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys

1075 1080 1085

Lys Met Lys Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr

1090 1095 1100

Gln Arg Lys Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser

1105 1110 1115 1120

Glu Leu Asp Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg

1125 1130 1135

Gln Ile Thr Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr

1140 1145 1150

Lys Tyr Asp Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr

1155 1160 1165

Leu Lys Ser Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr

1170 1175 1180

Lys Val Arg Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu

1185 1190 1195 1200

Asn Ala Val Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu

1205 1210 1215

Ser Glu Phe Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met

1220 1225 1230

Ile Ala Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe

1235 1240 1245

Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala

1250 1255 1260

Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr

1265 1270 1275 1280

Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val Arg Lys

1285 1290 1295

Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr Glu Val Gln

1300 1305 1310

Thr Gly Gly Phe Ser Lys Glu Ser Ile Arg Pro Lys Arg Asn Ser Asp

1315 1320 1325

Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly

1330 1335 1340

Phe Val Ser Pro Thr Val Ala Tyr Ser Val Leu Val Val Ala Lys Val

1345 1350 1355 1360

Glu Lys Gly Lys Ser Lys Lys Leu Lys Ser Val Lys Glu Leu Leu Gly

1365 1370 1375

Ile Thr Ile Met Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe

1380 1385 1390

Leu Glu Ala Lys Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys

1395 1400 1405

Leu Pro Lys Tyr Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg Met

1410 1415 1420

Leu Ala Ser Ala Arg Phe Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro

1425 1430 1435 1440

Ser Lys Tyr Val Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu

1445 1450 1455

Lys Gly Ser Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln

1460 1465 1470

His Lys His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser

1475 1480 1485

Lys Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala

1490 1495 1500

Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile

1505 1510 1515 1520

Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Arg Ala Phe Lys

1525 1530 1535

Tyr Phe Asp Thr Thr Ile Asp Arg Lys Val Tyr Arg Ser Thr Lys Glu

1540 1545 1550

Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr Gly Leu Tyr Glu

1555 1560 1565

Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp Ser Gly Gly Ser Gly

1570 1575 1580

Gly Ser Gly Gly Ser Thr Asn Leu Ser Asp Ile Ile Glu Lys Glu Thr

1585 1590 1595 1600

Gly Lys Gln Leu Val Ile Gln Glu Ser Ile Leu Met Leu Pro Glu Glu

1605 1610 1615

Val Glu Glu Val Ile Gly Asn Lys Pro Glu Ser Asp Ile Leu Val His

1620 1625 1630

Thr Ala Tyr Asp Glu Ser Thr Asp Glu Asn Val Met Leu Leu Thr Ser

1635 1640 1645

Asp Ala Pro Glu Tyr Lys Pro Trp Ala Leu Val Ile Gln Asp Ser Asn

1650 1655 1660

Gly Glu Asn Lys Ile Lys Met Leu Ser Gly Gly Ser Gly Gly Ser Gly

1665 1670 1675 1680

Gly Ser Thr Asn Leu Ser Asp Ile Ile Glu Lys Glu Thr Gly Lys Gln

1685 1690 1695

Leu Val Ile Gln Glu Ser Ile Leu Met Leu Pro Glu Glu Val Glu Glu

1700 1705 1710

Val Ile Gly Asn Lys Pro Glu Ser Asp Ile Leu Val His Thr Ala Tyr

1715 1720 1725

Asp Glu Ser Thr Asp Glu Asn Val Met Leu Leu Thr Ser Asp Ala Pro

1730 1735 1740

Glu Tyr Lys Pro Trp Ala Leu Val Ile Gln Asp Ser Asn Gly Glu Asn

1745 1750 1755 1760

Lys Ile Lys Met Leu Ser Gly Gly Ser Lys Arg Thr Ala Asp Gly Ser

1765 1770 1775

Glu Phe Glu Ser Pro Lys Lys Lys Arg Lys Val

1780 1785

<210> 6

atgaagagga cagccgacgg ctctgagttc gagtccccga agaagaagcg caaggtcttc 60

gagcgcaact acgacccacg cgagctgcgc aaagagacat acctcctcta cgagatcaag 120

tggggcaagt ccggaaagct ctggcgccat tggtgccaga acaacaggac acagcacgcc 180

gaggtgtact ttcttgagaa catcttcaac gcccgcaggt tcaacccgtc cacgcattgc 240

tcgatcacct ggtatctcag ctggtcccca tgcgccgagt gctcccaaaa gatcgtggac 300

ttcctcaaag agcacccgaa cgtcaacctc gagatctacg tggcccgcct ctactaccca 360

gagaacgaga gaaataggca gggcctccgc gacctcgtga attctggcgt gaccatccgc 420

atcatggacc tgccggacta caactactgc tggaaaacct tcgtgtccga ccaaggcggc 480

gacgaggatt attggccagg ccatttcgcc ccgtggatca agcagtactc cctcaaactt 540

tctggtggct ctagcggcgg ctcatctggc tctgagacac caggcacaag cgagtccgct 600

acgccagagt catctggtgg cagctccggc ggctccgaca agaagtactc catcggcctc 660

gctatcggca ccaacagcgt cggctgggcg gtgatcaccg acgagtacaa ggtcccgtcc 720

aagaagttca aggtcctggg caacaccgac cgccactcca tcaagaagaa cctcatcggc 780

gccctcctct tcgactccgg cgagacggcg gaggcgaccc gcctcaagcg caccgcccgc 840

cgccgctaca cccgccgcaa gaaccgcatc tgctacctcc aggagatctt ctccaacgag 900

atggcgaagg tcgacgactc cttcttccac cgcctcgagg agtccttcct cgtggaggag 960

gacaagaagc acgagcgcca ccccatcttc ggcaacatcg tcgacgaggt cgcctaccac 1020

gagaagtacc ccactatcta ccaccttcgt aagaagcttg ttgactctac tgataaggct 1080

gatcttcgtc tcatctacct tgctctcgct cacatgatca agttccgtgg tcacttcctt 1140

atcgagggtg accttaaccc tgataactcc gacgtggaca agctcttcat ccagctcgtc 1200

cagacctaca accagctctt cgaggagaac cctatcaacg cttccggtgt cgacgctaag 1260

gcgatccttt ccgctaggct ctccaagtcc aggcgtctcg agaacctcat cgcccagctc 1320

cctggtgaga agaagaacgg tcttttcggt aacctcatcg ctctctccct cggtctgacc 1380

cctaacttca agtccaactt cgacctcgct gaggacgcta agcttcagct ctccaaggat 1440

acctacgacg atgatctcga caacctcctc gctcagattg gagatcagta cgctgatctc 1500

ttccttgctg ctaagaacct ctccgatgct atcctccttt cggatatcct tagggttaac 1560

actgagatca ctaaggctcc tctttctgct tccatgatca agcgctacga cgagcaccac 1620

caggacctca ccctcctcaa ggctcttgtt cgtcagcagc tccccgagaa gtacaaggag 1680

atcttcttcg accagtccaa gaacggctac gccggttaca ttgacggtgg agctagccag 1740

gaggagttct acaagttcat caagccaatc cttgagaaga tggatggtac tgaggagctt 1800

ctcgttaagc ttaaccgtga ggacctcctt aggaagcaga ggactttcga taacggctct 1860

atccctcacc agatccacct tggtgagctt cacgccatcc ttcgtaggca ggaggacttc 1920

taccctttcc tcaaggacaa ccgtgagaag atcgagaaga tccttacttt ccgtattcct 1980

tactacgttg gtcctcttgc tcgtggtaac tcccgtttcg cttggatgac taggaagtcc 2040

gaggagacta tcaccccttg gaacttcgag gaggttgttg acaagggtgc ttccgcccag 2100

tccttcatcg agcgcatgac caacttcgac aagaacctcc ccaacgagaa ggtcctcccc 2160

aagcactccc tcctctacga gtacttcacg gtctacaacg agctcaccaa ggtcaagtac 2220

gtcaccgagg gtatgcgcaa gcctgccttc ctctccggcg agcagaagaa ggctatcgtt 2280

gacctcctct tcaagaccaa ccgcaaggtc accgtcaagc agctcaagga ggactacttc 2340

aagaagatcg agtgcttcga ctccgtcgag atcagcggcg ttgaggaccg tttcaacgct 2400

tctctcggta cctaccacga tctcctcaag atcatcaagg acaaggactt cctcgacaac 2460

gaggagaacg aggacatcct cgaggacatc gtcctcactc ttactctctt cgaggatagg 2520

gagatgatcg aggagaggct caagacttac gctcatctct tcgatgacaa ggttatgaag 2580

cagctcaagc gtcgccgtta caccggttgg ggtaggctct cccgcaagct catcaacggt 2640

atcagggata agcagagcgg caagactatc ctcgacttcc tcaagtctga tggtttcgct 2700

aacaggaact tcatgcagct catccacgat gactctctta ccttcaagga ggatattcag 2760

aaggctcagg tgtccggtca gggcgactct ctccacgagc acattgctaa ccttgctggt 2820

tcccctgcta tcaagaaggg catccttcag actgttaagg ttgtcgatga gcttgtcaag 2880

gttatgggtc gtcacaagcc tgagaacatc gtcatcgaga tggctcgtga gaaccagact 2940

acccagaagg gtcagaagaa ctcgagggag cgcatgaaga ggattgagga gggtatcaag 3000

gagcttggtt ctcagatcct taaggagcac cctgtcgaga acacccagct ccagaacgag 3060

aagctctacc tctactacct ccagaacggt agggatatgt acgttgacca ggagctcgac 3120

atcaacaggc tttctgacta cgacgtcgac cacattgttc ctcagtcttt ccttaaggat 3180

gactccatcg acaacaaggt cctcacgagg tccgacaaga acaggggtaa gtcggacaac 3240

gtcccttccg aggaggttgt caagaagatg aagaactact ggaggcagct tctcaacgct 3300

aagctcatta cccagaggaa gttcgacaac ctcacgaagg ctgagagggg tggcctttcc 3360

gagcttgaca aggctggttt catcaagagg cagcttgttg agacgaggca gattaccaag 3420

cacgttgctc agatcctcga ttctaggatg aacaccaagt acgacgagaa cgacaagctc 3480

atccgcgagg tcaaggtgat caccctcaag tccaagctcg tctccgactt ccgcaaggac 3540

ttccagttct acaaggtccg cgagatcaac aactaccacc acgctcacga tgcttacctt 3600

aacgctgtcg ttggtaccgc tcttatcaag aagtacccta agcttgagtc cgagttcgtc 3660

tacggtgact acaaggtcta cgacgttcgt aagatgatcg ccaagtccga gcaggagatc 3720

ggcaaggcca ccgccaagta cttcttctac tccaacatca tgaacttctt caagaccgag 3780

atcaccctcg ccaacggcga gatccgcaag cgccctctta tcgagacgaa cggtgagact 3840

ggtgagatcg tttgggacaa gggtcgcgac ttcgctactg ttcgcaaggt cctttctatg 3900

cctcaggtta acatcgtcaa gaagaccgag gtccagaccg gtggcttctc caaggagtct 3960

atccgtccaa agagaaactc ggacaagctc atcgctagga agaaggattg ggaccctaag 4020

aagtacggtg gtttcgtctc ccctactgtc gcctactccg tcctcgtggt cgccaaggtg 4080

gagaagggta agtcgaagaa gctcaagtcc gtcaaggagc tcctcggcat caccatcatg 4140

gagcgctcct ccttcgagaa gaacccgatc gacttcctcg aggccaaggg ctacaaggag 4200

gtcaagaagg acctcatcat caagctcccc aagtactctc ttttcgagct cgagaacggt 4260

cgtaagagga tgctggcttc cgctcgtttc ctccagaagg gtaacgagct tgctcttcct 4320

tccaagtacg tgaacttcct ctacctcgcc tcccactacg agaagctcaa gggttcccct 4380

gaggataacg agcagaagca gctcttcgtg gagcagcaca agcactacct cgacgagatc 4440

atcgagcaga tctccgagtt ctccaagcgc gtcatcctcg ctgacgctaa cctcgacaag 4500

gtcctctccg cctacaacaa gcaccgcgac aagcccatcc gcgagcaggc cgagaacatc 4560

atccacctct tcacgctcac gaacctcggc gcccctcgtg ctttcaagta cttcgacacc 4620

accatcgaca ggaaggttta caggtccacc aaggaggttc tcgacgctac tctcatccac 4680

cagtccatca ccggtcttta cgagactcgt atcgaccttt cccagcttgg tggtgattct 4740

ggtggatctg gcggaagcgg cggctctacc aatctctccg acatcatcga gaaagagaca 4800

ggcaagcagc tcgtgatcca agagtccatc ctcatgctcc cggaagaggt cgaggaagtg 4860

atcggcaaca agccagagtc cgacatcctc gtgcacaccg cctacgatga gtccaccgac 4920

gagaacgtga tgctcctcac ctctgacgcc ccagagtaca agccatgggc gctcgtgatt 4980

caggactcca acggcgagaa caagatcaag atgctctcag gcggcagcgg aggttcaggc 5040

ggctcaacaa acctcagcga tattattgag aaagaaaccg ggaagcaatt ggtcattcaa 5100

gagtcgattc tgatgttgcc cgaagaggtg gaagaggtta tcgggaacaa acccgagagc 5160

gacatcctgg tccatacggc gtatgacgag agcacggatg agaatgtcat gctcctgacc 5220

agcgacgcgc ccgagtataa gccttgggct cttgtcatcc aggacagcaa tggggaaaac 5280

aaaatcaaaa tgctgagcgg cggcagcaag aggacagctg atggctctga gttcgagtcc 5340

ccgaagaaga agcgcaaggt ctag 5364

<210> 7

ctcaccctgt tgtttggtgt tacttctgca gatgaagagg acagccg 47

<210> 8

gccgatggag tacttcttgt cggagccgcc ggagctgcca ccag 44

<210> 9

ctccggcggc tccgacaaga agtactccat c 31

<210> 10

ccagatccac cagaatcacc accaagctgg ga 32

<210> 11

cagcttggtg gtgattctgg tggatctggc ggaag 35

<210> 12

caaatgtttg aacgatcggg aggatcctag accttgcgct tcttc 45

<210> 13

acccccccac aggctcgcga gttttagagc tagaaat 37

<210> 14

tcgcgagcct gtgggggggt cggcagccaa gccagca 37

<210> 15

tcgtcggcgg cgatggtgag ttttagagct agaaat 36

<210> 16

tcaccatcgc cgccgacgac ggcagccaag ccagca 36

<210> 17

accgccaccg tcgtcgccaa gttttagagc tagaaat 37

<210> 18

ttggcgacga cggtggcggt caacacaagc ggcagc 36

<210> 19

tgcatgcatg cacccatgcg ttttagagct agaaat 36

<210> 20

gcatgggtgc atgcatgcat gccacggatc atctgc 36

<210> 21

atctctgcac tgaattgaat gttttagagc tagaaat 37

<210> 22

attcaattca gtgcagagat cggcagccaa gccagca 37

<210> 23

tccaccatgc accacgacgt gttttagagc tagaaat 37

<210> 24

acgtcgtggt gcatggtgga cggcagccaa gccagca 37

<210> 25

agctcaagct ccgcgccgcg ttttagagct agaaat 36

<210> 26

gcggcgcgga gcttgagctc aacacaagcg gcagc 35

<210> 27

atcagcgacc ggatctcccc gttttagagc tagaaat 37

<210> 28

ggggagatcc ggtcgctgat caacacaagc ggcagc 36

<210> 29

cattctccca gttcttcgcg ttttagagct agaaat 36

<210> 30

gcgaagaact gggagaatgt gccacggatc atctgc 36

<210> 31

ctccgtttta cctgtggaat cg 22

<210> 32

cggaggaaaa ttccatccac 20

<210> 33

ttcagaggtc tctaccgact agtcacgcgt atggaatcgg cagcaaa 47

<210> 34

agcgtgggtc tcgtcagggt ccatccactc caagctc 37

<210> 35

ttcagaggtc tctctgacac tggaatcggc agcaaagg 38

<210> 36

agcgtgggtc tcgtcttcac tccatccact ccaagctc 38

<210> 37

ttcagaggtc tctaagactt tggaatcggc agcaaagg 38

<210> 38

agcgtgggtc tcgagtcctt tccatccact ccaagctc 38

<210> 39

ttcagaggtc tctgactaca tggaatcggc agcaaagg 38

<210> 40

agcgtgggtc tcggtccaca tccatccact ccaagctc 38

<210> 41

ttcagaggtc tctggactag tggaatcggc agcaaagg 38

<210> 42

agcgtgggtc tcgcagatag tccatccact ccaagctc 38

<210> 43

ttcagaggtc tcttctgcaa tggaatcggc agcaaagg 38

<210> 44

agcgtgggtc tcgacctcaa tccatccact ccaagctc 38

<210> 45

ttcagaggtc tctaggtttc tggaatcggc agcaaagg 38

<210> 46

agcgtgggtc tcgagcgttc tccatccact ccaagctc 38

<210> 47

ttcagaggtc tctcgctgat tggaatcggc agcaaagg 38

<210> 48

agcgtgggtc tcgctcgacg cgtatccatc cactccaagc 40

<210> 49

gcggtgtcat ctatgttact ag 22

<210> 50

tgcaataact tcgtataggc t 21

Claims

1. A SpCas9 variant fusion protein, characterized by:

designated PeCBE-NG, comprising cytosine deaminase eCBE, a SpCas9 variant Cas9n-NG and a uracil glycosylase inhibitor protein;

2. The SpCas9 variant fusion protein of claim 1, characterized in that:

the SpCas9 variant fusion protein further comprises one or more of the following sequences: a linker, a nuclear localization signal, and amino acid residues or amino acid sequences introduced for the purpose of constructing a fusion protein, promoting the expression of a recombinant protein, obtaining a recombinant protein that is automatically secreted into the nucleus of a host cell, or facilitating the purification of a recombinant protein;

the joint is a flexible joint;

the amino acid sequence of the nuclear localization signal is shown as SEQ ID NO. 4;

the number of the nuclear localization signals is two, and the nuclear localization signals are respectively fused at the N end and the C end of the SpCas9 variant fusion protein;

the number of the uracil glycosylase inhibiting proteins is two, and the two uracil glycosylase inhibiting proteins are connected in series through a joint.

3. The SpCas9 variant fusion protein of claim 1, characterized in that: from N-terminal to C-terminal, the gene sequentially contains 1 nuclear localization signal, 1 cytosine deaminase eCBE, 1 SpCas9 variant Cas9N-NG, 2 serial uracil glycosylase inhibitor proteins and 1 nuclear localization signal.

4. The SpCas9 variant fusion protein of claim 1, characterized in that:

the full amino acid sequence of the SpCas9 variant fusion protein is shown as SEQ ID NO. 5.

5. A polynucleotide sequence characterized by: is a polynucleotide sequence encoding a SpCas9 variant fusion protein of any one of claims 1 to 4.

6. The polynucleotide sequence of claim 5, wherein: the polynucleotide sequence is shown in SEQ ID NO. 6.

7. A plant efficient cytosine single base editor, characterized in that: is obtained by integrating a polynucleotide sequence for encoding the SpCas9 variant fusion protein of any one of claims 1-4 into a plant transformation vector.

8. The plant efficient cytosine single base editor of claim 7, wherein:

the plant transformation vector is a binary expression vector, further is pCAMBIA1300 and a vector obtained by modifying the pCAMBIA1300 on the basis of the pCAMBIA1300, and further is pYLCRISPR/Cas9Pubi-H, and the polynucleotide sequence is inserted between Pst I and BamHI enzyme cutting sites of the vector.

9. The method for constructing a plant efficient cytosine single base editor as claimed in claim 7 or 8, comprising the steps of:

10. Use of a SpCas9 variant fusion protein of any one of claims 1 to 4, or a polynucleotide sequence of any one of claims 5 to 6, or a plant efficient cytosine single base editor of any one of claims 7 to 8 for single base editing of a plant genome.